Proton-Coupled Chromophore and Protein Structural Changes Control Phytochrome Activation.

Phytochromes are sensory photoreceptors in eukaryotes and prokaryotes that control physiological processes. In prototypical phytochromes, photoisomerization of the methine-bridged tetrapyrrole of the Pr state is the first step in (de)activating the photoreceptor. The underlying reaction sequence runs through a series of intermediate states. Among them, the Meta-Rc state plays a critical role since it precedes the formation of the Pfr state, which is linked to the functional secondary structure transition of the tongue, a phytochrome-specific peptide segment. In this work, we have studied the structure and reactions of Meta-Rc of the bacterial phytochrome Agp1 (Agrobacterium fabrum) by IR difference and resonance Raman spectroscopy. It is shown that the formation of Meta-Rc is associated with the enolization of the terminal ring D and the deprotonation of ring B or C, whereas reprotonation of the chromophore occurs with the decay of Meta-Rc. Proton migration represents the essential trigger for the secondary structure transition of the tongue since the β-sheet and α-helix structures can be interconverted by changing the pH. The pH-dependent conformational equilibrium is observed in Meta-Rc at 250 K and in Pfr at 290 K, albeit with different pKA values. The results show that the secondary structure transition is induced by chromophore-linked proton transfer steps rather than by conformational relaxations of the chromophore itself. In view of previous findings on the proton dependence of the reverse process in bathy phytochromes, we conclude that intramolecular proton transfer is an indispensable prerequisite for the secondary structure transition in phytochromes in general.